Microscope apparatus and program

ABSTRACT

Provided are a microscope apparatus capable of executing auto-focus control that appropriately follows a high-speed scan in a case of capturing a high-magnification and wide view image of an observation target contained in a container having large variations in the bottom surface, and a program. In a case where a microscope apparatus main body scans the bottom surface of the cultivation container by synchronously controlling a piezoelectric element and an actuator serving as optical axis-directional transport devices having different properties from each other, an objective lens of the imaging optical system is transported to a focus position in the optical axis direction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of PCT International Application No.PCT/JP2018/038979 filed on Oct. 19, 2018, which claims priority under 35U.S.C § 119(a) to Japanese Patent Application No. 2017-220179 filed onNov. 15, 2017. Each of the above application(s) is hereby expresslyincorporated by reference, in its entirety, into the presentapplication.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The technology of the present disclosure relates to a microscopeapparatus and a program.

2. Description of the Related Art

In the related art, a method for capturing an image of a multipotentialstem cell such as an embryonic stem (ES) cell or an induced pluripotentstem (iPS) cell, a differentiated and induced cell, or the like using amicroscope or the like, and capturing a feature of the image to decide adifferentiation state of the cell, or the like has been proposed.

The multipotential stem cell such as an ES cell or an iPS cell is ableto be differentiated into cells of various tissues, and may be appliedto regenerative medicine, development of medicines, explanation ofdiseases, or the like.

On the other hand, a high-magnification image can be obtained by imagingusing a microscope, but an observation region of an imaging opticalsystem of the microscope is limited to a narrow range. Therefore, inorder to obtain a high-magnification and wide view image of anobservation target, it has been proposed to perform a so-called tilingimaging. Tiling imaging is an imaging method for capturing a pluralityof narrow view images and connecting the captured narrow view images togenerate a wide view image. Specifically, it is possible to generate awide view image including an entire observation target by scanning anentire observation target by two-dimensionally moving the imagingoptical system of the microscope and a stage on which a cultivationcontainer such as a well plate containing the observation target isinstalled relative to each other, and connecting the images obtained foreach of the observation region.

SUMMARY OF THE INVENTION

In a case of performing the tiling imaging as described above, a focalposition of the imaging optical system is often set with reference to abottom surface of the cultivation container.

However, there is a millimeter-order fabrication tolerance in athickness of the bottom of the cultivation container. Moreover, in acase of performing high-magnification imaging, it is not possible toignore variations in the bottom surface of the cultivation container dueto this fabrication error. In other words, in a case of performinghigh-magnification imaging, it is not possible to assume that thethickness of the bottom of the cultivation container is uniform withrespect to each observation region in tiling imaging, and it isnecessary to measure the focal position for each observation region, andmove the imaging optical system to the focus position in an optical axisdirection for each observation region. Therefore, it is necessary tomove the imaging optical system of the microscope and the stage on whichthe cultivation container is installed relative to each other in athree-dimensional manner.

In order to move an objective lens of the imaging optical system to afocus position in an optical axis direction for each observation regionwith respect to variations in the bottom surface of the cultivationcontainer in a case of scanning the cultivation container at high speedusing the imaging optical system of the microscope, a mechanism ofmoving in a vertical direction at a high speed is preferable. Therefore,a piezoelectric element is used as an optical axis direction movingmember.

On the other hand, a drivable range of the piezoelectric element islimited. Therefore, the objective lens of the imaging optical systemcannot be moved in the optical axis direction beyond an upper limit atwhich the piezoelectric element may be deformed. Thus, in a case wherethere are large variations on the bottom surface of the cultivationcontainer, an optical axis direction moving mechanism using only thepiezoelectric element cannot perform an appropriate auto-focus control.

The microscope apparatus of JP2011-081211A comprises a vertical drivingunit including a stepping motor and a piezo element. However,JP2011-081211A does not disclose at all how to use the stepping motorand the piezo element in driving the objective lens in the verticaldirection.

With the technology according to JP2011-081211A, in a case where thereare large variations in the bottom surface of the cultivation container,it is difficult to perform an auto-focus control so that follow thehorizontal movement of the imaging optical system.

In view of the above-described problem, the technology of the presentdisclosure provides a microscope apparatus and a program capable ofperforming a focus control so as to follow the position of each regionin the optical axis direction according to a scanning situation.

According to a first aspect of the present disclosure, there is provideda microscope apparatus comprising: an imaging optical system capable offorming an image of observation target light indicating an observationtarget in a container in which the observation target is contained, onan imaging element; a drive source that includes a first moving membermovable along an optical axis direction of the imaging optical system,and a second moving member movable in the optical axis direction in arange wider than that of the first moving member and moves the imagingoptical system in the optical axis direction using the first movingmember and the second moving member; and a controller that controls thedrive source to cause the imaging element to form the image of theobservation target light in a focus state when the optical axis reachesa specific region, by moving the imaging optical system in the opticalaxis direction by using the first moving member and the second movingmember based on information on a distance in the optical axis directionbetween the specific region and a region to be imaged by the imagingelement at an imaging position, in a case where it is determined thatthe specific region exists at a position of an out-of-focus state in astate where the first moving member is moved to a limit of a movablerange of the first moving member before the optical axis reaches thespecific region among respective regions, in a state where the imagingelement scans the respective regions in the container by moving theimaging optical system with respect to the respective regions in thecontainer by a movement of at least one of a stage on which thecontainer is installed or the imaging optical system in a planeintersecting the optical axis direction.

According to a second aspect of the present disclosure, in the firstaspect, in the microscope apparatus, an amount of power in the opticalaxis direction distributed by the first moving member and the secondmoving member is determined in accordance with a relationship betweenthe information on the distance, information on a position of the secondmoving member in the optical axis direction before the specific regionis scanned, and information on a moving amount of the first movingmember.

According to a third aspect of the present disclosure, in the first orsecond aspect, in the microscope apparatus, the controller controls thedrive source to cause the imaging element to form the image of theobservation target light in the focus state when the optical axisreaches the specific region by applying the power in the optical axisdirection by only the first moving member out of the first moving memberand the second moving member to the imaging optical system based on theinformation on the distance, in a case where it is determined that thespecific region exists at a position of the focus state in a state wherethe first moving member is moved below the limit of the movable range,before the optical axis reaches the specific region among the respectiveregions in a state where the imaging element scans the respectiveregions in the container.

According to a fourth aspect of the present disclosure, in the thirdaspect, in the microscope apparatus, the power in the optical axisdirection by only the first moving member out of the first moving memberand the second moving member is determined in accordance with arelationship between the information on the distance and information ona moving amount of the first moving member.

According to a fifth aspect of the present disclosure, in the first tofourth aspects, in the microscope apparatus, the first moving member isa piezoelectric element that deforms along the optical axis direction ofthe imaging optical system.

According to a sixth aspect of the present disclosure, in the first tofifth aspects, in the microscope apparatus, the drive source includes apulse motor, and the second moving member moves in the optical axisdirection by receiving the power from the pulse motor.

According to a seventh aspect of the present disclosure, in the first tosixth aspects, in the microscope apparatus, the microscope apparatusfurther comprises a detection section that is provided along the opticalaxis direction and detects a position of the specific region in theoptical axis direction, where the controller controls the drive sourceto cause the imaging element to form the image of the observation targetlight in the focus state when the optical axis reaches the specificregion by applying the power in the optical axis direction by the firstmoving member and the second moving member to the imaging optical systembased on the information on the distance, in a case where a positiondetected by the detection section is the position of the out-of-focusstate in a state where the first moving member is moved to the limit ofthe movable range, before the optical axis reaches the specific regionamong the respective regions in a state where the imaging element scansthe respective regions in the container.

According to an eighth aspect of the present disclosure, in the seventhaspect, in the microscope apparatus, the detection section includes apair of sensors that are provided side by side with the imaging opticalsystem interposed therebetween in a main scanning direction with respectto the respective regions, and that detect the position of the specificregion in the optical axis direction respectively, and the controllercontrols the drive source to cause the imaging element to form the imageof the observation target light in the focus state when the optical axisreaches the specific region by applying the power in the optical axisdirection by the first moving member and the second moving member to theimaging optical system based on the information on the distance, in acase where a position detected by a sensor of the pair of sensors thatreaches the specific region in the main scanning direction earlier isthe position of the out-of-focus state in a state where the first movingmember is moved to the limit of the movable range, before the opticalaxis reaches the specific region among the respective regions in a statewhere the imaging element scans the respective regions in the container.

According to a ninth aspect of the present disclosure, in the seventh oreighth aspect, the microscope apparatus further comprises a holdingmember that holds the drive source, where the detection section is heldby the holding member.

According to a tenth aspect of the present disclosure, in the first toninth aspects, in the microscope apparatus, the imaging optical systemhas an objective lens movable in the optical axis direction, and theobjective lens is moved in the optical axis direction by the firstmoving member and the second moving member.

According to an eleventh aspect of the present disclosure, in the firstto tenth aspects, the container is a well plate having a plurality ofwells.

According to a twelfth aspect of the present disclosure, there isprovided a program causing a computer to function as the controllerincluded in the microscope apparatus according to the first to eleventhaspects.

According to the technology of the present disclosure, it is possible tocause the focus control to follow the position of each region in theoptical axis direction in accordance with a scanning situation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic configuration diagram showing an example of aconfiguration of a microscope apparatus according to a first embodiment.

FIG. 2 is a schematic diagram showing an example of a configuration ofan imaging optical system included in the microscope apparatus accordingto the first embodiment.

FIG. 3 is a schematic configuration diagram showing an example of aconfiguration of an imaging optical system driving section included inthe microscope apparatus according to the first embodiment.

FIG. 4 is a schematic configuration diagram showing an example of aconfiguration of a stage included in the microscope apparatus accordingto the first embodiment.

FIG. 5 is a block diagram showing an example of a hardware configurationof an electric system of the microscope apparatus according to the firstembodiment.

FIG. 6 is a block diagram showing a main configuration of a part relatedto a technology of the present disclosure of a microscope apparatus mainbody and a microscope control device included in the microscopeapparatus according to the first embodiment.

FIG. 7 is a conceptual diagram showing an example of a scanning positionof an observation target region in a cultivation container installed ona stage of the microscope apparatus according to the first embodiment.

FIG. 8A is a conceptual diagram showing a first form example indicatinga positional relationship of an imaging optical system, first and seconddisplacement sensors, and a cultivation container in a case where anobservation target region is located at an optional position in acultivation container installed on a stage of the microscope apparatusaccording to the first embodiment.

FIG. 8B is a conceptual diagram showing a second form example indicatinga positional relationship of an imaging optical system, first and seconddisplacement sensors, and a cultivation container in a case where anobservation target region is located at an optional position in acultivation container installed on a stage of the microscope apparatusaccording to the first embodiment.

FIG. 9 is a state transition diagram for explaining an example of atiming of an auto-focus control in the microscope apparatus according tothe first embodiment.

FIG. 10 is a flowchart showing an example of a flow of wide view imageacquisition processing according to the first embodiment.

FIG. 11 is a flowchart showing an example of a flow of stage movingprocessing according to the first embodiment.

FIG. 12 is a conceptual diagram showing an example of a relationshipbetween an observation target region and a distance in a Z direction.

FIG. 13 is a flowchart showing an example of a flow of focus controlpreparation processing according to the first embodiment.

FIG. 14 is a conceptual diagram showing an example of a pulse profilecreated by executing focus control preparation processing according tothe first embodiment.

FIG. 15 is a schematic configuration diagram showing an example of avoltage table according to the first embodiment.

FIG. 16 is a flowchart showing an example of a flow of piezoelectricelement control processing according to the first embodiment.

FIG. 17 is a flowchart showing an example of a flow of motor controlprocessing according to the first embodiment.

FIG. 18 is a schematic configuration diagram showing an example of aconfiguration of a microscope apparatus according to a secondembodiment.

FIG. 19 is a schematic configuration diagram showing an example of aconfiguration of a detection section included in the microscopeapparatus according to the second embodiment.

FIG. 20 is a diagram provided to explain switching of a position of adisplacement sensor in a detection section included in the microscopeapparatus according to the second embodiment.

FIG. 21 is a schematic configuration diagram showing a modificationexample of a configuration of an imaging optical system driving sectionaccording to the first embodiment.

FIG. 22 is a conceptual diagram showing an example of a mode in which awide view image acquisition program is installed in a microscopeapparatus from a storage medium storing a wide view image acquisitionprogram according to the first and second embodiments.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, details of each embodiment of the present disclosure willbe described with reference to the drawings.

First Embodiment

<Overview of System>

Hereinafter, a microscope apparatus 90 according to a first embodimentof the technology of the present disclosure will be described in detailwith reference to the drawings. FIG. 1 is a block diagram showing aschematic configuration of a microscope apparatus 90 according to afirst embodiment.

The microscope apparatus 90 includes a microscope apparatus main body 10and a microscope control device 20. The microscope apparatus 90 may beconnected to a display device 30 and an input device 40 via themicroscope control device 20.

The microscope apparatus 90 is an example of the microscope apparatus ofthe present disclosure.

The microscope apparatus main body 10 captures a phase difference imageof cultured cells to be observed by the microscope apparatus 90.Specifically, as shown in FIG. 1 as an example, the microscope apparatusmain body 10 comprises a white light source 11 that emits white light, acondenser lens 12, a slit plate 13, an imaging optical system 14, animaging optical system driving section 15, an imaging element 16, and adetection section 18.

The imaging element 16 is an example of an imaging element of thepresent disclosure. The imaging optical system 14 is an example of animaging optical system of the present disclosure.

The slit plate 13 is one obtained by providing a ring-shaped slit thattransmits white light with respect to a light-shielding plate thatshields white light emitted from the white light source 11, and thewhite light passes through the slit to form a ring-shaped illuminationlight L.

As shown in FIG. 2 as an example, the imaging optical system 14comprises a phase difference lens 14 a and an imaging lens 14 d. Thephase difference lens 14 a comprises an objective lens 14 b and a phaseplate 14 c. The phase plate 14 c has a configuration in which a phasering is formed in a transparent plate that is transparent with respectto a wavelength of the illumination light L. The size of the slit of theabove-described slit plate 13 is in a cooperative relationship with thephase ring of the phase plate 14 c.

The phase ring has a configuration in which a phase membrane that shiftsa phase of incident light by ¼ of a wavelength and a light-reducingfilter that reduces incident light are formed in a ring shape. The phaseof direct light incident onto the phase ring shifts by ¼ of a wavelengthafter passing through the phase ring, and its brightness is weakened. Onthe other hand, most of diffracted light diffracted by an observationtarget passes through the transparent plate of the phase plate 14 c, andits phase and brightness are not changed.

The phase difference lens 14 a having the objective lens 14 b is movedin an optical axis direction of the objective lens 14 b by the imagingoptical system driving section 15 shown in FIG. 1, for example. In thefirst embodiment, the optical axis direction of the objective lens 14 band a Z direction (vertical direction) are the same direction. Anauto-focus control is performed as the phase difference lens 14 a ismoved so as to match the focus position in the Z direction, and contrastof a phase difference image captured by the imaging element 16 isadjusted.

Here, in the first embodiment, the Z-directional position of the bottomsurface of a cultivation container 50 described later, which ispreviously installed on a stage 51 described later, is detected and setas a reference plane. Then, a reference position in the Z direction isset for the imaging optical system 14 so as to be in a focus positionwith respect to the reference plane.

In a case where a boundary surface between the bottom of the cultivationcontainer 50, that is, the bottom of the cultivation container 50(described later) containing the observation target and the observationtarget matches the reference plane, the imaging optical system 14 placedat the reference position should be in a focus state with respect to theobservation target. However, the bottom surface of the cultivationcontainer 50 is close to the reference plane but is not completelymatched. This is because there are variations on the bottom surface ofthe cultivation container 50 due to a fabrication error or the like.That is, the actual focus position with respect to each region on thebottom surface of the cultivation container 50 often does not match thereference position.

The auto-focus control according to the present disclosure is to correcta deviation between the respective regions on the bottom surface of thecultivation container 50 and the reference plane by moving the imagingoptical system 14 in the Z direction, and control so that the imagingoptical system 14 is actually in a focus position with respect to theobservation target in each region.

Further, a configuration in which a magnification of the phasedifference lens 14 a is changeable may be used. Specifically, aconfiguration in which the phase difference lenses 14 a or the imagingoptical systems 14 having different magnifications are interchangeablemay be used. The interchange between the phase difference lens 14 a andthe imaging optical systems 14 may be automatically performed, or may bemanually performed by a user.

As shown in FIG. 3 as an example, the imaging optical system drivingsection 15 includes an actuator 15B and a piezoelectric element 15Adisposed vertically above the actuator 15B. The actuator 15B isconnected to a pulse motor M as an example. The piezoelectric element15A and the actuator 15B function as a Z-directional transport devicefor moving the objective lens 14 b of the imaging optical system 14 inthe Z direction, based on a control signal output from a focuscontroller 21 described later.

The imaging optical system driving section 15 is an example of a drivesource of the present disclosure. The piezoelectric element 15A is anexample of a first moving member of the present disclosure, and theactuator 15B is an example of a second moving member of the presentdisclosure.

In a case where the piezoelectric element 15A as the Z-directionaltransport device and the actuator 15B are compared, there are thefollowing differences. The piezoelectric element 15A can move theimaging optical system 14 in the Z direction at a higher speed than theactuator 15B. On the other hand, a drivable range of the piezoelectricelement 15A is smaller than that of the actuator 15B.

The imaging optical system driving section 15 is configured to causephase difference light passed through the phase difference lens 14 a topass through as it is.

Further, the drive source of the actuator 15B is not limited to thepulse motor, but may be any drive source having a sufficiently largedrivable range as compared with the piezoelectric element 15A. As adrive source of the actuator 15B, a solenoid or other knownconfiguration can be used. Moreover, the imaging optical system drivingsection 15 may be configured by a combination of a first piezoelectricelement having a small drivable range and a second piezoelectric elementhaving a large drivable range or a combination of a first actuatordriven by a small pulse motor and a second actuator driven by a largepulse motor.

The imaging lens 14 d receives phase difference light that has passedthrough the phase difference lens 14 a and the imaging optical systemdriving section 15, and forms an image on the imaging element 16.

The imaging element 16 captures an image on the basis of the phasedifference image formed by the imaging lens 14 d. As the imaging element16, a charge-coupled device (CCD) image sensor, a complementarymetal-oxide semiconductor (CMOS) image sensor, or the like may be used.As the imaging element, an imaging element in which color filters ofred, green, and blue (R, G, and B) are provided may be used, or amonochromic imaging element may be used.

The detection section 18 detects the position of the cultivationcontainer 50 installed on the stage 51 in the Z direction (verticaldirection). The detection section 18 specifically comprises a firstdisplacement sensor 18 a and a second displacement sensor 18 b. Thefirst displacement sensor 18 a and the second displacement sensor 18 bin the first embodiment are laser displacement meters that irradiate thecultivation container 50 with laser light and detect the reflected lightto detect the Z-directional position of the bottom surface of thecultivation container 50. In the present disclosure, the bottom surfaceof the cultivation container 50 refers to a boundary surface between thebottom of the cultivation container 50 and cells to be observed, thatis, an observation target installation surface.

The cultivation container 50 is an example of a container of the presentdisclosure.

In the first embodiment, the first displacement sensor 18 a and thesecond displacement sensor 18 b are provided side by side in an Xdirection shown in FIG. 1 with the phase difference lens 14 a interposedtherebetween (see FIGS. 8A and 8B).

As an example, each of the first displacement sensor 18 a and the seconddisplacement sensor 18 b is disposed so as to be separated from thephase difference lens 14 a by a distance nine times the one side D ofthe square observation target region R, that is, by 9D. The observationtarget region R will be described later.

Information on the Z-directional position of the cultivation container50 detected by the detection section 18 is output to a focus controller21 described later (see FIG. 6), and the focus controller 21 controlsthe imaging optical system driving section 15 based on the inputposition information to perform the auto-focus control. The detection ofthe position of the cultivation container 50 by the first displacementsensor 18 a and the second displacement sensor 18 b and the auto-focuscontrol by the focus controller 21 will be described later.

Between the slit plate 13, and the phase difference lens 14 a and thedetection section 18, the stage 51 is provided. On the stage 51, thecultivation container 50 in which cells that are observation targets arecontained is installed.

In the microscope apparatus according to the first embodiment, as shownin FIG. 7 as an example, a well plate having six wells W is used as thecultivation container 50. However, the cultivation container 50 is notlimited to this. For example, a well plate having 24 or 96 wells can beused as the cultivation container 50, and a well plate having anoptional number of wells or a container other than a well plate such asa petri dish or dish can be used. An appropriate container can beselected as the cultivation container 50 depending on the observationtarget and the purpose of the observation.

Further, examples of cells contained in the cultivation container 50include multipotential stem cells such as induced pluripotent stem (iPS)cells and embryonic stem (ES) cells, cells of nerve induced anddifferentiated from stem cells, skin, myocardium, and liver, and cellsof skin, retina, myocardium, blood cells, nerve, and organ extractedfrom the human body.

The stage 51 is moved in the X direction and a Y direction orthogonal toeach other by a stage driving device 17 (see FIGS. 1 and 6) describedlater. The X direction and the Y direction are directions orthogonal tothe Z direction, and are directions orthogonal to each other in ahorizontal plane. In the first embodiment, the X direction is a mainscanning direction, and the Y direction is a sub scanning direction.

As shown in FIG. 4 as an example, a rectangular opening 51 a is formedin a center of the stage 51. The cultivation container 50 is installedon a member forming the opening 51 a, and phase difference light ofcells in the cultivation container 50 passes through the opening 51 a.

The display device 30 displays a composite phase difference imagegenerated and stored in wide view image acquisition processing describedlater, and comprises, for example, a liquid crystal display. Further,the display device 30 may be configured by a touch panel, and may alsobe used as the input device 40.

The input device 40 comprises a mouse, a keyboard, and the like, andreceives various setting inputs by the user. The input device 40receives setting inputs such as an instruction to change themagnification of the phase difference lens 14 a and an instruction tochange the moving velocity of the stage.

Next, the configuration of the microscope control device 20 thatcontrols the microscope apparatus main body 10 will be described. FIG. 5shows an example of a configuration of an electric system of themicroscope control device 20.

The microscope control device 20 comprises a computer. The computer ofthe microscope control device 20 comprises a central processing unit(CPU) 70, a primary storage section 72, a secondary storage section 74,an input/output interface (I/O) 76, and the like. The CPU 70, theprimary storage section 72, the secondary storage section 74, and theI/O 76 are connected by a bus line.

The CPU 70 controls the entire microscope apparatus. The primary storagesection 72 is a volatile memory used as a work area or the like in acase of executing various programs. An example of the primary storagesection 72 includes a random access memory (RAM). The secondary storagesection 74 is a non-volatile memory in which various programs andvarious parameters are stored in advance, and stores a wide view imageacquisition program 80 which is an example of a program according to thetechnology of the present disclosure. Examples of the secondary storagesection 74 include an electrically erasable programmable read-onlymemory (EEPROM), a flash memory, and the like. The I/O 76 controlstransmission and reception of various information between the microscopeapparatus main body 10 and the microscope control device 20.

The CPU 70 reads out the wide view image acquisition program 80 from thesecondary storage section 74. Then, the CPU 70 develops the read wideview image acquisition program 80 in the primary storage section 72 andexecutes the developed wide view image acquisition program 80, therebyoperating as the focus controller 21 and the stage controller 22illustrated in FIG. 6 as an example.

The stage controller 22 controls the stage driving device 17 to move thestage 51 in the X direction and the Y direction. The stage drivingdevice 17 is, for example, an actuator having a piezoelectric element orthe like.

The focus controller 21 performs control (auto-focus control) on theimaging optical system driving section 15 based on the information onthe Z-directional position of the cultivation container 50 detected bythe detection section 18. The auto-focus control is realized by drivingthe imaging optical system driving section 15 and moving the objectivelens 14 b of the imaging optical system 14 in the optical axisdirection.

The focus controller 21 is an example of a controller according to thetechnology of the present disclosure. The focus controller 21 controlsthe imaging optical system driving section 15 to cause the imagingelement 16 to form the image of the observation target light(observation light indicating the observation target in the cultivationcontainer 50) in a focus state when the optical axis of the imagingoptical system 14 reaches an observation scheduled region by applyingthe power in the Z direction by the piezoelectric element 15A and theactuator 15B to the imaging optical system 14, based on information onthe distance in the optical axis direction between the region (that is,the region to be imaged by the imaging element at the imaging position)currently being imaged by the imaging element and the observationscheduled region in a case where a predetermined condition is satisfiedin a state where the imaging element is scanning each region in thecultivation container 50. The state in which the imaging element scansrespective regions in the cultivation container 50 means a state inwhich the stage 51 is moved in the XY plane by the X direction and the Ydirection to move the imaging element with respect to each observationregion in the cultivation container 50 such that the imaging element 16scans the respective regions in the cultivation container 50. Further,the predetermined condition refers to a condition that, the observationscheduled region exists at a position of an out-of-focus state in theoptical axis direction in a state where the piezoelectric element 15A isdeformed by a limit amount of the deformation amount of thepiezoelectric element 15A.

<Wide View Image Acquisition Processing>

Hereinafter, the process of acquiring a wide view image by themicroscope apparatus 90 according to the first embodiment of the presentdisclosure will be described in detail.

FIGS. 10, 11, 13, 16, and 17 are flowcharts illustrating an example of aprogram for performing the wide view image acquisition processing in thefirst embodiment. FIGS. 10, 11, 13, 16, and 17 show an example of aprogram executed by the microscope control device 20.

Here, the program executed by the microscope control device 20 isspecifically executed by the CPU 70 of the microscope control device 20functioning as the focus controller 21 and the stage controller 22.

In the following, for the convenience of explanation, the wide viewimage acquisition processing will be described in three parts of (1)start of a flow, (2) scanning processing, and (3) processing after thescanning processing.

[Start of Flow]

In the microscope apparatus 90 according to the first embodiment,scanning of the observation target is performed by the microscopeapparatus main body 10 continuously capturing a narrow view image of thecultivation container 50 disposed on the stage 51 while the microscopecontrol device 20 performs two-dimensional movement control on the stage51 and focus control on the imaging optical system 14.

First, a user who desires to capture a wide view image of an observationtarget installs the cultivation container 50 containing cells to beobserved on the stage 51.

As described above, in the first embodiment, as an example, adescription will be given assuming that the cultivation container 50 isa well plate having six wells W.

In a case where the user instructs the microscope control device 20 tocapture a wide view image through the input device 40, the wide viewimage acquisition processing according to the first embodiment isstarted. Hereinafter, the CPU 70 of the microscope control device 20reads out the wide view image acquisition program 80 from the secondarystorage section 74 and executes the processing. FIG. 10 is a flowchartshowing the entire processing.

[Scanning Processing]

In step S100, CPU 70 executes scanning processing. In step S100, stagemoving processing (see FIG. 12), focus control preparation processing(see FIG. 13), piezoelectric element control processing (see FIG. 16),motor control processing (see FIG. 17), and continuous imaging of anarrow view image by the microscope apparatus main body 10 are executed.

The stage moving processing, the focus control preparation processing,the piezoelectric element control processing, the motor controlprocessing, and the continuous imaging of the narrow view image by themicroscope apparatus main body 10 are performed in synchronization witheach other. Hereinafter, the processing will be described on the premiseof this synchronous control.

(Stage Moving Processing Subroutine)

The stage moving processing included in step S100 in FIG. 10 is shown asa subroutine in FIG. 11. FIG. 11 is an example of a processing programexecuted by the CPU 70 of the microscope control device 20 functioningas the stage controller 22.

In step S202, the stage controller 22 performs an initial setting of theX-axis movement direction for the stage 51. As an example, the X-axismovement direction of the stage 51 is set to a negative direction.

Next, in step S204, the stage controller 22 causes the stage drivingdevice 17 to start moving the stage 51 along the set X-axis movementdirection. Immediately after the start of the subroutine in FIG. 11,since the X-axis movement direction is set to the negative direction instep S202, the stage 51 starts to move in the X-axis negative direction.In this case, as shown in FIG. 8A, the imaging optical system 14 of thestationary microscope apparatus main body 10 moves relatively to thestage 51 in the X-axis positive direction. In the following, therelative movement of the imaging optical system 14 (and the observationtarget region R) with respect to the stage 51 due to the movement of thestage 51 by the stage driving device 17 is referred to as the movementof the imaging optical system 14 (and the observation target region R)for the convenience of explanation.

FIGS. 8A and 8B are diagrams showing positional relationships on the XYplane between the imaging optical system 14 in a case where theobservation target region R is located at an optional position on thebottom surface of cultivation container 50, the first displacementsensor 18 a and the second displacement sensor 18 b, and the cultivationcontainer 50. The observation target region R is a region on the bottomsurface of the cultivation container 50 where the microscope apparatusmain body 10 can capture an image to generate a phase difference image.With the relative movement of the imaging optical system 14 with respectto the stage 51, the observation target region R also relatively moveswith respect to the stage 51. Hereinafter, the traveling direction ofthe imaging optical system 14 and the observation target region R on theplane including the bottom surface of the cultivation container 50accompanying the X-axis directional movement of the stage 51 is referredto as the X-axis scanning direction. Further, the XY plane including thebottom surface of the cultivation container 50 is referred to as ascanning plane.

Hereinafter, the observation target region R is assumed to be a squareregion for the convenience of explanation, but is not limited to this.

Note that, the microscope apparatus main body 10 comprises a horizontalposition detection sensor not shown in FIG. 1. The stage controller 22detects the current position of the observation target region R on thescanning plane in the stage 51 using the horizontal position detectionsensor.

In the first embodiment, the observation target region R moves at auniform velocity along the X-axis scanning direction.

In a case where the observation target region R of the imaging opticalsystem 14 reaches an end point position on the scanning plane, thedetermination as to whether the observation target region R has reachedthe end point position in step S206 is positive. Here, the end pointposition is a position at which scanning in the X-axis direction on thescanning plane ends, and is shown in FIG. 7.

On the other hand, the stage controller 22 continues to move the stage51 in the X-axis movement direction by the stage driving device 17 untilthe determination in step S206 is positive. That is, the imaging opticalsystem 14 continues to relatively move in the X-axis scanning directionwith respect to the stage 51 until the determination in step S206 ispositive.

In the process in which the observation target region R moves in theX-axis scanning direction, the CPU 70 of the microscope control device20 causes the microscope apparatus main body 10 to capture an image of aregion overlapping the observation target region R on the bottom surfaceof the cultivation container 50 to generate a plurality of phasedifference images. That is, a phase difference image of each region onthe bottom surface of the cultivation container 50 that is continuousalong the X-axis scanning direction is generated.

Here, the focus control processing described later is executed insynchronization with the stage moving processing, and therefore it isnoted that, in each region on the bottom surface of the cultivationcontainer 50 superimposed in the observation target region R, theZ-directional position of the objective lens 14 b of the imaging opticalsystem 14 is set to the focus position. Accordingly, in the process inwhich the observation target region R relatively moves in the X-axisscanning direction, a phase difference image of a focus state isgenerated for each region on the bottom surface of the cultivationcontainer 50 continuous in the X-axis scanning direction.

Further, the CPU 70 of the microscope control device 20 stores theplurality of generated phase difference images in the primary storagesection 72, for example. Note that, the plurality of generated phasedifference images may be stored in a cache memory (not shown) of the CPU70 or the secondary storage section 74.

In a case where the determination in step S206 is positive, the stagecontroller 22 ends the movement of the stage 51 along the X-axisscanning direction by the stage driving device 17 in step S208. Further,the CPU 70 of the microscope control device 20 ends the continuouscapturing of the narrow view image by the microscope apparatus main body10. Next, the processing proceeds to step S210.

In step S210, the stage controller 22 determines whether the observationtarget region R has reached a scanning end point E. The X-axis scanningend point E is a point at which the scanning processing ends on thebottom surface of the cultivation container 50, and is exemplarily shownin FIG. 6.

In a case where the determination in step S210 is negative, theprocessing proceeds to step S212.

In step S212, the stage controller 22 causes the stage driving device 17to move the stage 51 by one unit in the Y-axis negative direction. Here,the one unit refers to a distance corresponding to a length D of oneside of the observation target region R. In this regard, reference canbe made to FIG. 7. Therefore, on the bottom surface of the cultivationcontainer 50, the observation target region R moves by one unit in theY-axis positive direction. Next, the processing proceeds to step S214.

In step S214, the stage controller 22 reverses the X-axis movementdirection with respect to the stage 51. Thereby, the X-axis scanningdirection of the observation target region R is reversed. Then, theprocessing of FIG. 11 returns to step S204, the stage controller 22starts the X-axis scanning again, and the CPU 70 of the microscopecontrol device 20 resumes the continuous capturing of the narrow viewimage by the microscope apparatus main body 10.

For example, in a case where step S214 is executed for the first time inthe processing of FIG. 11, since the X-axis movement direction withrespect to the stage 51 is set to the negative direction in step S202,the X-axis movement direction with respect to the stage 51 is set to thepositive direction as a result of the reverse. In this case, in a casewhere the processing in FIG. 11 returns to step S204, the stage 51starts to move in the X-axis positive direction. Accordingly, in thiscase, as shown in FIG. 8B, the imaging optical system 14 (and theobservation target region R) of the stationary microscope apparatus mainbody 10 is relatively moved in the X-axis negative direction withrespect to the stage 51.

The processing in FIG. 11 is continued until the determination in stepS210 is positive.

As described above, in a case where the stage controller 22 executes thestage moving processing subroutine, the stage 51 is moved in the Xdirection and the Y direction, the observation target region R of theimaging optical system 14 scans the bottom surface of the cultivationcontainer 50 two-dimensionally, and a phase difference image of eachregion is generated and stored.

The solid line M in FIG. 7 shows an example of the movement of theobservation target region R on the scanning plane including the bottomsurface of the cultivation container 50 in the scanning processing.

As shown in FIG. 7, the observation target region R of the imagingoptical system 14 moves along the solid line M from a scanning startpoint S to a scanning end point E. That is, the observation targetregion R scans in the X-axis positive direction (rightward in FIG. 7),moves in the Y-axis positive direction (downward in FIG. 7), and furtherscans in the X-axis negative direction (leftward in FIG. 7). Next, theobservation target region R is moved again in the Y-axis positivedirection and scans again in the X-axis positive direction. As describedabove, the observation target region R scans the bottom surface of thecultivation container 50 two-dimensionally by repeatedly performing thereciprocating movement in the X direction and the movement in the Ydirection.

The following points should be noted regarding the setting of the endpoint position and the stage moving velocity in the stage movingprocessing.

In the first embodiment, in order to perform scanning on the entirebottom surface of the cultivation container 50, as shown in FIG. 7, itis necessary to relatively move the imaging optical system 14, the firstdisplacement sensor 18 a, and the second displacement sensor 18 b to theranges R1 and R2 outside the range of the cultivation container 50 inthe X direction. For the width of the range R1 in the X direction, it isnecessary to ensure at least the distance between the first displacementsensor 18 a and the imaging optical system 14 in the X direction, andfor the width of the range R2 in the X direction, it is necessary toensure at least the distance between the second displacement sensor 18 band the imaging optical system 14 in the X direction. In order to reducethe scanning time of the observation target region R as much aspossible, it is desirable to make the scanning range of the observationtarget region R as narrow as possible. Therefore, it is desirable thatthe width of the range R1 in the X direction is the distance between thefirst displacement sensor 18 a and the imaging optical system 14 in theX direction, and it is desirable that the width of the range R2 in the Xdirection is the distance between the second displacement sensor 18 band the imaging optical system 14 in the X direction.

On the other hand, in a case where the observation target region R isscanned in the range of the cultivation container 50 by moving the stage51 in the X direction, it is desirable that the moving velocity of theobservation target region R in the range of the cultivation container 50is uniform. Accordingly, when the movement of the stage 51 in the Xdirection starts, it is necessary to accelerate the stage 51 to reach acertain velocity, and when the movement of the stage 51 in the Xdirection ends, it is necessary to decelerate the stage 51 from thecertain velocity for stopping.

Further, in a case where the moving velocity of the stage 51 in the Xdirection is to be the certain velocity, it is possible to rapidlycontrol such that the moving velocity becomes the certain velocitywithout making an acceleration region, but in a case where such acontrol is performed, a liquid level of a culture medium or the likecontained in the cultivation container 50 together with cells shakes,which may cause lowering in image quality of a phase difference image.In addition, in a case where the stage 51 is stopped, the same problemmay occur.

Accordingly, in the first embodiment, the range R1 and the range R2shown in FIG. 7 are set to acceleration/deceleration regions for thestage 51 to move in the X direction. By setting theacceleration/deceleration regions on both sides of the range of thecultivation container 50 in the X direction in this way, it is possibleto scan the observation target region R at a uniform velocity in therange of the cultivation container 50, without uselessly enlarging ascanning range. Further, it is possible to prevent the above-describedshake of the liquid level of the culture medium.

(Focus Control Processing Subroutine)

The focus control processing included in step S100 of FIG. 10 is shownas a subroutine in FIGS. 13, 16, and 17.

As described above, in the first embodiment, the first displacementsensor 18 a and the second displacement sensor 18 b are provided side byside in the X direction with the imaging optical system 14 interposedtherebetween as shown in FIGS. 8A and 8B. Therefore, in the stage movingprocessing described above, in a case where the observation targetregion R of the imaging optical system 14 scans each region on thebottom surface of the cultivation container 50 continuous along the Xdirection, the detection section 18 can detect the Z-directionalposition of the bottom surface of the cultivation container 50 withrespect to the region positioned forward of the position of theobservation target region R in the X-axis scanning direction.

Specifically, in a case where the observation target region R is movingin the direction of the arrow shown in FIG. 8A (rightward in FIG. 8A),among the first displacement sensor 18 a and the second displacementsensor 18 b, the first displacement sensor 18 a positioned forward ofthe observation target region R in the X-axis scanning direction detectsthe Z-directional position of the bottom surface of the cultivationcontainer 50. In a case where the observation target region R furthermoves to the right in FIG. 8A, in each region on the bottom surface ofthe cultivation container 50 along the X-axis scanning direction, theZ-directional position of the objective lens 14 b of the imaging opticalsystem 14 can be adjusted to the focus position by executing focuscontrol based on parameters including the Z-directional position of thebottom surface of the cultivation container 50 detected by the firstdisplacement sensor 18 a and the height of the reference plane of thecultivation container 50 detected in advance. That is, by executing thefocus control, in a case where the observation target region R moves tothe right in FIG. 7, each region can be captured while keeping theimaging optical system 14 in a focus state with respect to the bottomsurface of the cultivation container 50.

On the other hand, in a case where the observation target region R ismoving in the direction of the arrow shown in FIG. 8B (leftward in FIG.8B), among the first displacement sensor 18 a and the seconddisplacement sensor 18 b, the second displacement sensor 18 b positionedforward of the observation target region R in the X-axis scanningdirection detects the Z-directional position of the bottom surface ofthe cultivation container 50. Then, as described with reference to FIG.8A, by executing the focus control, in a case where the observationtarget region R moves to the left in FIG. 8B, each region can becaptured while keeping the imaging optical system 14 in a focus statewith respect to the bottom surface of the cultivation container 50.

Thus, by switching the Z-directional position detection of the bottomsurface of the cultivation container 50 using the first displacementsensor 18 a and the Z-directional position detection of the bottomsurface of the cultivation container 50 using the second displacementsensor 18 b in accordance with the movement direction of the observationtarget region R, prior to capturing the observation target region R, theposition information of the cultivation container 50 in the Z directionin the position of the observation target region R can always beobtained.

The focus controller 21 performs a focus control by adjusting theZ-directional position of the objective lens 14 b of the imaging opticalsystem 14 by using the imaging optical system driving section 15including the piezoelectric element 15A and the actuator 15B.Specifically, the focus controller 21 executes the focus control bycontrolling the amount of voltage applied to the piezoelectric element15A and the pulse input to the pulse motor M for driving the actuator15B.

Normally, the focus controller 21 executes focus control processing byusing only the piezoelectric element 15A. As described above, thepiezoelectric element 15A can move the imaging optical system 14 in theZ direction at a higher speed than the actuator 15B. However, since thepiezoelectric element 15A has a smaller drivable range than the actuator15B, a situation may occur in which the objective lens 14 b cannot bemoved a required distance only by the piezoelectric element 15A.

Description will be made regarding this with reference to FIG. 12. Forconvenience, the following description is based on the assumption thatthe observation target region R moves in the X-axis positive directionas shown in FIG. 8A.

For convenience of explanation, it is assumed that the X-axis directionof the bottom surface of the cultivation container 50 is divided by oneside D of the observation target region R. In a case where theobservation target region R is in the I-th region, the observationscheduled region is disposed in the (I+10)-th region. That is, theobservation scheduled region is positioned in a region ten units aheadof the observation target region R in the X-axis scanning direction.FIG. 9 is a diagram showing the above relationship.

Here, the observation scheduled region is a region on the bottom surfaceof the cultivation container 50 positioned vertically above the firstdisplacement sensor 18 a, and is a region where the observation targetregion R is overlapped after a predetermined time is elapsed. Asdescribed above, as an example, the first displacement sensor 18 a isdisposed so as to be separated from the phase difference lens 14 a inthe X direction by a distance that is nine times the one side D of theobservation target region R.

The observation scheduled region is an example of a specific region inthe present disclosure.

FIG. 12 is a diagram showing the focus position in each region from theI-th region to (I+10)-th region shown in FIG. 9. In FIG. 12, theobservation target region R is currently positioned in the I-th region,and the observation scheduled region is the (I+10)-th region.

In FIG. 12, the lower dashed line represents the height (theZ-directional position of the objective lens 14 b at the focus positionwith respect to the reference plane) corresponding to the referenceplane of the bottom surface of the cultivation container 50, the centerdashed line represents the current Z-directional position of theobjective lens 14 b, and the upper dashed line represents the upperlimit of the drivable range of the piezoelectric element 15A. Further,horizontal bars represent the focus positions of the respective regionsfrom the I-th region to the (I+10)-th region. In the preceding scan, theZ-directional position of the bottom surface of the cultivationcontainer 50 has already been detected for each of the I-th to (I+10)-thregions by the first displacement sensor 18 a. The focus position ineach region can be derived by subtracting the detected Z-directionalposition of each region from the Z-directional position of thecultivation container 50 on the reference plane.

The observation target region R moves each region in the X-axis scanningdirection from the I-th region to the (I+1)-th region, the (I+2)-thregion, and the like to the (I+10)-th region (that is, the observationscheduled region) by synchronized stage moving processing. The focuscontroller 21 controls the imaging optical system driving section 15 toadjust the Z-directional position of the objective lens 14 b in eachregion to the respective focus position.

However, in the situation shown in FIG. 12, the difference between thefocus position of the (I+10)-th region (that is, the observationscheduled region) and the focus position (that is, the lower dashed lineof FIG. 12) corresponding to the reference plane exceeds a threshold.This situation is called a range over of the piezoelectric element 15A.The threshold is a value determined based on the drivable range of thepiezoelectric element 15A, and is, for example, an upper limit of amovable distance of the piezoelectric element 15A. The threshold is anexample of a limit of a movable range of the first moving memberaccording to the present disclosure.

Therefore, the focus controller 21 cannot cause the imaging opticalsystem 14 to reach the focus position at the time point when theobservation target region R reaches the observation scheduled region, bythe control using only the piezoelectric element 15A.

Therefore, the focus controller 21 executes the focus control by drivingthe actuator 15B in addition to the piezoelectric element 15A. Asdescribed above, the actuator 15B has a larger drivable range than thepiezoelectric element 15A. Therefore, by driving the piezoelectricelement 15A and the actuator 15B in synchronization with each other, themovement of the distance exceeding the threshold becomes possible.

Although not shown in FIG. 12, the objective lens 14 b can betransported vertically below the reference plane by appropriatelydisposing the piezoelectric element 15A in the Z direction. That is, itis not necessary to match the lowest position where the piezoelectricelement 15A is deformed in the Z direction to the position correspondingto the reference plane (lower dashed line in FIG. 12), and it ispossible to set the lower limit of the drivable range below thereference plane in the Z direction.

Synchronous control of the piezoelectric element 15A and the actuator15B can be appropriately performed by using various optimizationcalculation technologies. In the optimization calculation, it isparticularly necessary to consider that the actuator 15B has a lowerZ-directional moving velocity than the piezoelectric element 15A.

Hereinafter, as an example, an example of a case where the synchronouscontrol of the piezoelectric element 15A and the actuator 15B isrealized by using the pulse profile 400 (see FIG. 14) and the voltagetable 500 (see FIG. 15) will be described.

The focus control processing according to the first embodiment includesa focus control preparation processing subroutine shown in FIG. 13, apiezoelectric element control processing subroutine shown in FIG. 16,and a motor control processing subroutine shown in FIG. 17. The focuscontroller 21 performs synchronous control of the piezoelectric element15A and the actuator 15B by synchronizing and executing the focuscontrol preparation processing subroutine, the piezoelectric elementcontrol processing subroutine, and the motor control processingsubroutine.

In the focus control preparation processing subroutine, a pulse profilefor executing motor control is generated and adjusted. In thepiezoelectric element control processing subroutine, the piezoelectricelement 15A is controlled based on the voltage table (which canfluctuate based on a pulse profile). In the motor control processingsubroutine, the pulse motor M that drives the actuator 15B is controlledbased on the pulse profile.

First, the focus control preparation processing subroutine of FIG. 13will be described. For convenience, description will be made using asituation where the observation target region R is located at the I-thregion as shown in FIG. 12.

In step S302, the focus controller 21 detects the Z-directional positionof the observation scheduled region on the bottom surface of thecultivation container 50 by the first displacement sensor 18 a.

Next, in step S304, the focus controller 21 stores the detectedinformation on the Z-directional position in the storage device. As anexample, the Z-directional position is stored in a cache memory (notshown) of the CPU 70. Further, the detected Z-directional position maybe stored in the primary storage section 72 or the secondary storagesection 74.

Next, in step S306, the focus controller 21 derives a distance that is adifference in the Z direction between the focus position (that is, thelower dashed line in FIG. 12) corresponding to the reference plane andthe focus position of (I+10)-th region (that is, observation scheduledregion).

The calculated distance is recorded in a cache memory (not shown) of theCPU 70 as an example. Further, the distance may be stored in the primarystorage section 72 or the secondary storage section 74.

In order to appropriately capture the observation scheduled region, itis necessary to move the imaging optical system 14 by a predetermineddistance in the Z direction in a case where the observation targetregion R moves to the current observation scheduled region.Specifically, the predetermined distance is a value obtained bysubtracting the current Z-directional position of the observation targetregion R from the distance derived in step S306.

Then, in step S308, the focus controller 21 determines whether thedistance is smaller than a threshold. As described above, the thresholdis, for example, the upper limit of the movable distance of thepiezoelectric element 15A.

In a case where the determination in step S308 is positive, theprocessing of FIG. 13 proceeds to step S310. In step S310, the focuscontroller 21 sets a flag (turns on the flag) for the observationscheduled region. The flag indicates that the focus position of theobservation scheduled region can be reached only by the piezoelectricelement 15A. The flag is recorded in a cache memory (not shown) of theCPU 70 as an example.

Next, in step S312, the focus controller 21 determines whether a pulseprofile has been created. The details of the pulse profile will bedescribed later.

In a case where a pulse profile exists, the focus controller 21 adjuststhe pulse profile in step S314 based on the information on theZ-directional position of the (I+10)-th region stored in step S304. In acase where there is no pulse profile, the processing of step S314 is notexecuted.

Then, in a case where the observation target region R moves to theadjacent (I+1)-th region by the synchronized stage moving processing,the determination in step S322 is positive, and the processing in FIG.13 proceeds to step S324. In this case, the observation target region Ris positioned in the (I+1)-th region, and the (I+11)-th region is a newobservation scheduled region.

In step S324, the focus controller 21 determines whether the newobservation scheduled region has reached the scanning end point in thestage moving processing executed by the stage controller 22. Thedetermination in step S324 corresponds to step S210 of the stage movingprocessing subroutine shown in FIG. 11.

In a case where the determination in step S324 is negative, theprocessing in FIG. 13 returns to step S302 again, and new processing isstarted. That is, for the (I+1)-th region where the observation targetregion R is currently disposed, the (I+11)-th region is set as a newobservation scheduled region, and the processing in step S302 andprocessing subsequent to step S302 are executed. In this way, while thestage controller 22 scans the bottom surface of the cultivationcontainer 50, the focus controller 21 continues the focus controlpreparation processing.

In a case where the determination in step S308 is negative, in stepS316, the focus controller 21 determines whether a pulse profile hasbeen created.

In a case where a pulse profile exists, the focus controller 21 adjuststhe pulse profile in step S318 based on the information on theZ-directional position of the (I+10)-th region stored in step S304.

On the other hand, in a case where the pulse profile does not exist, instep S320, the focus controller 21 generates a pulse profile based onthe information on the Z-directional position of each of the I-th to(I+10)-th regions stored in the preceding processing and the pulse motorperformance value such as the Z-directional moving velocity. The pulseprofile is recorded in a cache memory (not shown) of the CPU 70, forexample.

Then, in a case where the observation target region R moves to theadjacent (I+1)-th region by the synchronized stage moving processing,the determination in step S322 is positive, and the processing in FIG.13 proceeds to step S324. Subsequent processing is the same as in a casewhere the determination in step S308 is positive.

Hereinafter, the pulse profile will be described in detail. FIG. 14shows an example of the pulse profile 400. The pulse profile representsa functional relationship between the time and the number of pulses perunit time input to the pulse motor M. The pulse profile 400 defines thenumber of pulses per unit time to be input to the pulse motor M, fromthe current time (t=0) to the time when it reaches the region ten unitsahead in the X-axis scanning direction. The pulse motor M that drivesthe actuator 15B is controlled based on the pulse profile 400.Specifically, the number of pulses input to the pulse motor M while theobservation target region R moves by one unit from the current positionto the region in the X-axis scanning direction is determined based onthe time required for the observation target region R to move by oneunit from the current position to the region in the X-axis scanningdirection and the corresponding number of pulses per unit time.

The pulse profile 400 fluctuates every time the observation targetregion R moves by one unit to the region in the X-axis scanningdirection. As an example, the solid curve in FIG. 14 represents thepulse profile 400 in a case where the observation target region R is inthe I-th region, and the dashed curve represents the pulse profile 400in a case where the observation target region R is in the (I+1)-thregion.

With the fluctuation of the pulse profile 400, the number of pulsesinput to the pulse motor M also changes while the observation targetregion R moves by one unit from the current position to the region inthe X-axis scanning direction. As described above, the pulse profile 400defines the number of pulses per unit time to be input to the pulsemotor M to the time when it reaches the region ten units ahead in theX-axis scanning direction. However, it should be noted that the numberof pulses actually determined to be input to the pulse motor M isdetermined only by the number of pulses per unit time corresponding tothe time required for the observation target region R to move by oneunit from the current position to the region in the X-axis scanningdirection.

As described above, the pulse profile 400 is generated based on theinformation on the Z-directional position of each region from thecurrent position to the region ten units ahead and parameters such asthe performance value of the pulse motor, and is adjusted over time.

In the synchronous control of the piezoelectric element 15A and theactuator 15B according to the first embodiment, the actuator 15B iscontrolled based on the pulse profile 400. On the other hand, thepiezoelectric element 15A is controlled based on the voltage table 500.

FIG. 15 shows an example of the voltage table 500. The voltage table 500includes information on the voltage to be applied to the piezoelectricelement 15A with respect to the displacement amount in the Z direction.

As an example, a situation (where the function in FIG. 14 matches thehorizontal axis) can be considered in which a pulse profile is notcreated or the number of pulses is zero at all times in the pulseprofile. Each voltage value shown in the voltage table 500 in thissituation is called a reference value.

In this case, the actuator 15B is not driven, and the objective lens 14b of the imaging optical system 14 is moved in the Z direction only bythe piezoelectric element 15A. In a case where the displacement amountto transport the objective lens 14 b in the Z direction is Z3, the focuscontroller 21 refers to the voltage table 500 and derives that thevoltage value to be applied to the piezoelectric element 15A is V3.

In a case where the objective lens 14 b of the imaging optical system 14is moved in the Z direction only by the piezoelectric element 15A, forexample, in a case where the observation target region R moves one unitfrom the current position to the region in the X-axis scanningdirection, assuming that the distance in the Z direction between thefocus position of the current position (I-th region) and the focusposition of the next position ((I+1)-th region) is Z3, the objectivelens 14 b can be adjusted to the focus position in the (I+1)-th regionby applying the reference value V3 to the piezoelectric element 15A.

On the other hand, a case can be considered in which the pulse profile400 exists and the number of pulses per unit time during the time whenthe observation target region R moves by one unit from the currentposition to the region in the X-axis scanning direction is not zero. Inthis case, the actuator 15B is driven, and the objective lens 14 b ofthe imaging optical system 14 moves in the Z direction together with thepiezoelectric element 15A. In this case, the voltage value of thevoltage table 500 is changed based on the pulse profile 400. That is,since the actuator 15B moves in the Z direction based on the pulseprofile 400, even though the displacement amount to transport theobjective lens 14 b in the Z direction is Z3, the voltage value to beapplied to the piezoelectric element 15A is not the same as V3 that isthe previous reference value. By applying the voltage valuecorresponding to the displacement amount obtained by subtracting thedisplacement amount by the actuator 15B from Z3 to the piezoelectricelement 15A, the objective lens 14 b of the imaging optical system 14 istransported to the focus position.

Based on the above, processing in FIG. 16 will be described. Forconvenience, description will be made using a situation where theobservation target region R is located at the I-th region as shown inFIG. 12.

In a case where the capturing of the phase difference image in theobservation target region R ends, a positive determination is made instep S602, and the processing in FIG. 16 proceeds to step S604.

In step S604, the focus controller 21 acquires the information on theZ-directional position of the (I+10)-th region from the I-th region(that is, the current position of the observation target region R)stored in the preceding focus control preparation processing. Asdescribed above, as an example, the information on the Z-directionalposition is stored in a cache memory (not shown) of the CPU 70.

Next, in step S606, the focus controller 21 determines whether the flagis off for a region ten units ahead in the X-axis scanning directionfrom the current position of the observation target region R (that is,the observation scheduled region). As described above, the flag isstored in a cache memory (not shown) of the CPU 70 as an example in thepreceding focus control preparation processing.

If the determination in step S606 is negative, the focus position of the(I+10)-th region (that is, the observation scheduled region) fallswithin the drivable range of the piezoelectric element 15A. Next, instep S608, the focus controller 21 determines whether the number ofpulses from the current time (t=0) of the pulse profile to the time whenthe observation target region R has moved by one unit to the region inthe X-axis scanning direction is zero. In a case where no pulse profilehas been created, the number of pulses is assumed to be zero.

In a case where the determination in step S608 is positive, the focusposition of the observation scheduled region falls within the drivablerange of the piezoelectric element 15A, and the displacement by theactuator 15B is not performed. In this case, each voltage value of thevoltage table 500 shown in FIG. 15 is kept at the reference value.

Then, in a case where the observation target region R reaches theadjacent (I+1)-th region by the synchronized stage moving processing,the determination in step S612 is positive. In this case, since thedisplacement by the actuator 15B is not performed, in step S614, thefocus controller 21 applies the reference value of the voltage table 500to the piezoelectric element 15A in the (I+1)-th region. As a result, inthe (I+1)-th region, the objective lens 14 b of the imaging opticalsystem 14 is moved to the focus position by using only the piezoelectricelement 15A.

Next, a case can be considered in which the determination in step S606is negative and the determination in step S608 is negative. In thiscase, the focus position of the observation scheduled region fallswithin the drivable range of the piezoelectric element 15A, but theactuator 15B has already been driven.

Therefore, in step S610, the focus controller 21 adjusts the voltagetable 500 based on the pulse profile 400. As a result, the voltage table500 is changed from the reference value.

Then, in a case where the observation target region R reaches theadjacent (I+1)-th region by the synchronized stage moving processing,the determination in step S612 is positive. In this case, in step S614,the focus controller 21 applies a voltage of a value corrected from thereference value of the voltage table 500 to the piezoelectric element15A in the (I+1)-th region. Further, the actuator 15B is displaced inthe Z direction based on the pulse profile by the synchronized motorcontrol processing described later. As a result, the objective lens 14 bof the imaging optical system 14 is moved to the focus position in the(I+1)-th region by the displacement of the piezoelectric element 15A andthe displacement of the actuator 15B.

Further, a case can be considered in which the determination in stepS606 is positive. In this case, the focus position of the observationscheduled region exceeds the drivable range of the piezoelectric element15A.

In step S610, the focus controller 21 adjusts the voltage table 500based on the pulse profile 400. Then, the focus controller 21 executesthe processing in steps S612 and S614 in the same manner as in a casewhere the determination in step S606 is negative and the determinationin step S608 is negative.

In a case where the processing in step S614 ends, the processing in FIG.16 proceeds to step S616. In step S616, the focus controller 21determines whether the observation target region R has reached thescanning end point in the stage moving processing executed by the stagecontroller 22. The determination in step S616 corresponds to step S210of the stage moving processing subroutine shown in FIG. 11.

In a case where the determination in step S616 is negative, theprocessing in FIG. 16 returns to step S602, and the above processing isrepeated again. In this way, while the stage controller 22 is executingthe stage moving processing, the focus controller 21 continues theprocessing of applying a voltage to the piezoelectric element based onthe voltage table 500.

Next, the motor control processing will be described. FIG. 17 shows amotor control processing subroutine executed by the focus controller 21.

Once the pulse profile is generated in the focus control preparationprocessing shown in FIG. 13, the motor control processing subroutine isstarted.

In step S702, the focus controller 21 controls the number of pulsesinput to the pulse motor M that drives the actuator 15B based on thepulse profile 400. The actuator 15B is displaced in the Z directionbased on the number of input pulses. The processing in step S702 iscontinued until the stage moving processing executed by the stagecontroller 22 ends.

It should be noted that, in step S702, in a case where the number ofpulses at the current time (t=0) of the pulse profile 400 is zero, nopulse is input to the pulse motor M and the actuator 15B is not driven.

In the stage moving processing executed by the stage controller 22, in acase where the observation target region R reaches the scanning endpoint, the determination in step S704 is positive, and the motor controlprocessing subroutine ends. The determination in step S704 correspondsto the determination in step S210 of the stage moving processingsubroutine shown in FIG. 11 and the determination in step S616 of thepiezoelectric element control processing subroutine shown in FIG. 16.

As described above, as an example of the focus control using thepiezoelectric element 15A and the actuator 15B together, the entiresynchronous control of the piezoelectric element 15A and the actuator15B using the pulse profile 400 and the voltage table 500 has beendescribed. Hereinafter, a typical example of the synchronous controlwill be described.

As described above, usually, the focus controller 21 executes the focuscontrol processing using only the piezoelectric element 15A.

In this state, in the processing in FIG. 13, the positive determinationin step S308, the negative determination in step S312, and the negativedetermination in step S324 are repeated, and the pulse profile 400 isnot generated.

In this case, in the processing in FIG. 16, a negative determination instep S606, and a positive determination in step S608 are repeated.Therefore, in the processing in FIG. 16, each voltage value of thevoltage table 500 is not changed from the reference value. That is, inthe piezoelectric element control processing in FIG. 16, the focuscontroller 21 repeatedly applies the voltage of the reference value tothe piezoelectric element 15A.

On the other hand, since the pulse profile 400 is not generated in theprocessing in FIG. 13, the motor control processing in FIG. 17 is notstarted.

In this way, the focus control processing is performed only by thepiezoelectric element 15A.

Next, it is assumed that the focus position of the observation scheduledregion exceeds the drivable range of the piezoelectric element 15A at acertain time point in the stage moving processing executed by the stagecontroller 22. In this case, in the processing in FIG. 13, a negativedetermination is made in step S308 and a negative determination is madein step S316, and the pulse profile 400 is generated in step S320.

In a case where the pulse profile 400 is generated, the motor controlprocessing in FIG. 17 is started, and the focus control processing inwhich the piezoelectric element 15A and the actuator 15B aresynchronized is executed.

In this process, the pulse profile fluctuates with time in step S314 orstep S318 in FIG. 13. On the other hand, in step S610 of FIG. 16, eachvoltage value of the voltage table 500 fluctuates from the referencevalue and changes with time.

In this manner, the focus control processing using both thepiezoelectric element 15A and the actuator 15B is performed.

Next, it is assumed that the stage moving processing has progressed andthe bottom surface of the cultivation container 50 has passed a regionhaving large variations. In this case, a positive determination is madein step S308 and a positive determination is made in step S312 in FIG.13, and it is considered that the pulse profile is adjusted to a valueclose to zero in step S314.

In a case where the number of pulses becomes zero at all times in thepulse profile (in a case where the function in FIG. 14 matches thehorizontal axis), no pulse is input to the pulse motor M in S702 in FIG.17, and the actuator 15B stops. On the other hand, a negativedetermination is made in step S606 and a positive determination is madein step S608 in FIG. 16, and each voltage value in the voltage table 500does not fluctuate from the reference value.

The return to the normal state is performed in this way, and the focuscontrol processing using only the piezoelectric element 15A is executedagain.

As described above, the control using the pulse profile 400 and thevoltage table 500 has been described as an example of the focus controlfor synchronously controlling the piezoelectric element 15A and theactuator 15B. However, the focus control for synchronously controllingthe piezoelectric element 15A and the actuator 15B is not limited to thecontrol using the pulse profile 400 and the voltage table 500. Byapplying various known optimization technologies, it is possible toappropriately control the piezoelectric element 15A and the actuator 15Band execute the focus control of the first embodiment.

In the first embodiment, since the Z-directional position of thecultivation container 50 is detected in advance for each observationtarget region R as described above, the timing at which the position ofthe cultivation container 50 in each observation target region R isdetected and the timing at which the phase difference image is capturedare temporally shifted. Therefore, the movement of the imaging opticalsystem 14 (the objective lens 14 b) in the Z direction, that is, theauto-focus control is performed after the position of the cultivationcontainer 50 is detected by the first displacement sensor 18 a or thesecond displacement sensor 18 b and before the observation target regionR reaches the detection position.

Here, in a case where the timing of the auto-focus control is too early,the Z-directional position of the cultivation container 50 may beshifted, and the focus position may be shifted after the auto-focuscontrol and before the observation target region R reaches the detectionposition for some reason.

Therefore, it is desirable that the timing of the auto-focus control isa timing immediately before the observation target region R reaches thedetection position and a timing at which the phase difference image iscaptured in the detection position in time. For example, as shown inFIG. 9, in a case where the observation target region R sequentiallymoves in the X direction and the detection position by the detectionsection 18 is the position of Pd indicated by oblique lines, it isdesirable that the time immediately before the observation target regionR reaches the detection position is a period from the time when theobservation target region R passes the position Pr of the observationtarget region R adjacent to the detection position Pd to when theobservation target region R reaches the detection position Pd. Theauto-focus control may be performed when the observation target region Rreaches the detection position Pd.

In the first embodiment, the time from the timing of detection by thefirst displacement sensor 18 a or second displacement sensor 18 b to thetiming of auto-focus control using the position information of thedetection position is preset so that the timing of the auto-focuscontrol is the desired timing as described above.

In a case where the moving velocity of the stage 51 is changed by, forexample, changing the magnification of the phase difference lens 14 a,the above-described preset time may be changed according to the changein the moving velocity of the stage 51. Alternatively, in a case wherethe moving velocity of the stage 51 is changed instead of changing thepreset time described above, the distance between the first displacementsensor 18 a or the second displacement sensor 18 b and the imagingoptical system 14 may be changed by moving the first displacement sensor18 a or the second displacement sensor 18 b in the X direction.

[Processing after Scanning Processing]

When the scanning processing in step S100 shown in FIG. 10 ends, thenarrow view image in the focus state is generated for each region on thebottom surface of the cultivation container 50, and stored in theprimary storage section 72, the cache memory (not shown) of the CPU 70,or the secondary storage section 74.

The processing shown in FIG. 10 proceeds to step S102.

In step S102, the CPU 70 of the microscope control device 20 reads outand combines the stored narrow view images to generate one compositephase difference image (that is, a wide view image) showing the entirebottom surface of the cultivation container 50.

Next, in step S104, the CPU 70 of the microscope control device 20stores the generated composite phase difference image, and ends the wideview image acquisition processing. The generated composite phasedifference image can be stored in, for example, the secondary storagesection 74.

Note that, the stored wide view image can be displayed on the displaydevice 30.

According to the first embodiment, it is possible to cause the focuscontrol to follow the position of each region in the optical axisdirection according to a scanning situation.

Specifically, by synchronously controlling the piezoelectric element andthe actuator in the Z direction connected to the pulse motor, even in acase where there are large variations on the bottom surface of thecultivation container, the auto-focus control can be performedappropriately during high-speed scanning of the entire bottom surface ofthe cultivation container. The focus control for each region can bespeeded up according to the position of each region on the bottomsurface of the cultivation container in the optical direction, and as aresult, the scanning time of the entire bottom surface of thecultivation container can be reduced.

Second Embodiment

Next, a microscope apparatus according to a second embodiment of thetechnology of the present disclosure will be described in detail withreference to the drawings. The microscope apparatus according to thesecond embodiment differs from the microscope apparatus according to thefirst embodiment in the configuration of the detection section. Sincethe other configuration of the microscope apparatus of the secondembodiment is the same as that of the first embodiment, the followingdescription will be given focusing on the configuration of the detectionsection of the microscope apparatus of the second embodiment.

The detection section 18 of the first embodiment comprises twodisplacement sensors 18 a and 18 b, and switches the displacement sensorto be used according to the change of the observation target region R inthe X-axis scanning direction. On the other hand, a detection section 19of the second embodiment includes one displacement sensor, and switchesthe position of the displacement sensor according to the change of theobservation target region R in the X-axis scanning direction.

FIGS. 19 and 20 are diagrams showing a specific configuration of thedetection section 19 of the second embodiment. As shown in FIGS. 19 and20, the detection section 19 comprises a displacement sensor 19 a and aguide mechanism 19 b that guides the displacement sensor 19 a to movethe position of the displacement sensor 19 a.

The displacement sensor 19 a is the same as the first and seconddisplacement sensors 18 a and 18 b of the first embodiment. That is, thedisplacement sensor 19 a is configured of a laser displacement sensor.

The guide mechanism 19 b comprises a semicircular arc-shaped guidemember, so that the displacement sensor 19 a is moved along the guidemember. The guide member moves the displacement sensor 19 a from oneside to the other side in the X direction with the imaging opticalsystem 14 (objective lens 14 b) being interposed therebetween.

FIG. 19 is a diagram showing a position of the displacement sensor 19 ain a case where the X-axis scanning direction of the observation targetregion R is an arrow direction in FIG. 19 (rightward in FIG. 19). On theother hand, FIG. 20 is a diagram showing a position of the displacementsensor 19 a in a case where the X-axis scanning direction of theobservation target region R is an arrow direction in FIG. 20 (leftwardin FIG. 20). In a case where the X-axis scanning direction of theobservation target region R is changed to the arrow direction in FIG. 20from the arrow direction in FIG. 19, the displacement sensor 19 a ismoved from the position shown in FIG. 19 along the guide member of theguide mechanism 19 b, and is switched to the position shown in FIG. 20.

In the second embodiment, the above-described guide mechanism 19 b isprovided as a displacement sensor moving mechanism for moving theposition of the displacement sensor, but the configuration of thedisplacement sensor moving mechanism is not limited thereto, and otherconfigurations may be used as long as the position of the displacementsensor is capable of being similarly changed.

The other configurations and operations of the microscope apparatus ofthe second embodiment are the same as in the microscope apparatus of thefirst embodiment.

MODIFICATION EXAMPLE

Hereinafter, a modification example of the present disclosure will bedescribed. Hereinafter, the present modification example will bedescribed based on the first embodiment, but the present modificationexample can also be applied to the second embodiment.

FIG. 21 shows an example in which a modification example is applied toan imaging optical system driving section 15 including a piezoelectricelement 15A and an actuator 15B.

In the imaging optical system driving section 15 of the first embodimentshown in FIG. 3, the piezoelectric element 15A is disposed on theactuator 15B.

In the modification example of FIG. 21, the piezoelectric element 15A isheld by the holding member 24. Further, the actuator 15B is disposed ona side surface of the holding member 24. In the present modificationexample, the actuator 15B transmits power for moving the holding member24 in the Z direction to the holding member 24 via the side surface ofthe holding member 24. The objective lens 14 b of the imaging opticalsystem 14 to be held by the piezoelectric element 15A held by theholding member 24 moves in the Z direction by the holding member 24moving in the Z direction by the actuator 15B.

The positional relationship between the piezoelectric element 15A andthe actuator 15B is not limited to the modification example shown inFIG. 21. It is sufficient as long as the objective lens 14 b can beappropriately transported in the Z direction.

In the first and second embodiments, a configuration in which theobservation target region R is scanned by moving the stage 51 is shown,but the disclosure is not limited thereto. For example, a configurationin which the stage 51 is fixed and the observation target region R isscanned by moving the imaging optical system 14 and a differentconfiguration relating to the capturing of a phase difference image maybe used. A configuration in which the observation target region R isscanned by moving all of the stage 51 and the imaging optical system 14and the other configuration relating to the capturing of the differentphase difference image may be used.

Further, in the first and second embodiments, the disclosure is appliedto a phase contrast microscope, but the disclosure is not limited to thephase contrast microscope, and may be applied to a different microscopesuch as a differential interference microscope or a bright fieldmicroscope.

In addition, in the first and second embodiments, a configuration inwhich a phase difference image formed by the imaging optical system 14is captured by the imaging element 16 is shown, but a configuration inwhich an imaging element is not provided and an observation opticalsystem or the like is provided so that a user is able to directlyobserve a phase difference image of an observation target formed by theimaging optical system 14 may be used.

Further, in the first and second embodiments, a configuration in whichthe wide view image acquisition program 80 is read out from thesecondary storage section 74 has been described as an example. However,it is not always necessary to store the program in the secondary storagesection 74 from the beginning. For example, as shown in FIG. 22, thewide view image acquisition program 80 may first be stored in anyportable storage medium 800 such as a Solid State Drive (SSD), aUniversal Serial Bus (USB) memory, or a Digital Versatile Disc-Read OnlyMemory (DVD-ROM). In this case, the wide view image acquisition program80 in the storage medium 800 is installed in the microscope apparatus90, and the installed wide view image acquisition program 80 is executedby the CPU 70.

The wide view image acquisition program 80 may be stored in a storagesection such as another computer or a server connected to the microscopeapparatus 90 via a communication network (not shown), and the wide viewimage acquisition program 80 may be downloaded in response to a requestfrom the microscope apparatus 90. In this case, the downloaded wide viewimage acquisition program 80 is executed by the CPU 70.

The wide view image acquisition processing described in the first andsecond embodiments is merely an example. Therefore, needless to say,unnecessary steps may be deleted, new steps may be added, or theprocessing order may be changed without departing from the scope of theinvention.

In the first and second embodiments, a configuration in which wide viewimage acquisition processing is realized by the software configurationusing a computer has been described as an example. However, thetechnology of the present disclosure is not limited to this. Forexample, the wide view image acquisition processing may be executed onlyby a hardware configuration such as a Field-Programmable Gate Array(FPGA) or an Application Specific Integrated Circuit (ASIC) instead of asoftware configuration using a computer. The wide view image acquisitionprocessing may be executed by a configuration in which a softwareconfiguration and a hardware configuration are combined.

EXPLANATION OF REFERENCES

10: microscope apparatus main body

11: white light source

12: condenser lens

13: slit plate

14: imaging optical system

14 a: phase difference lens

14 b: objective lens

14 c: phase plate

14 d: imaging lens

15: imaging optical system driving section

15A: piezoelectric element

15B: actuator

16: imaging element

17: stage driving device

18: detection section

18 a: displacement sensor

18 b: displacement sensor

19: detection section

19 a: displacement sensor

19 b: guide mechanism

20: microscope control device

21: focus controller

22: stage controller

24: holding member

30: display device

40: input device

50: cultivation container

5: stage

51 a: opening

72: primary storage section

74: secondary storage section

80: wide view image acquisition program

90: microscope apparatus

400: pulse profile

500: voltage table

800: storage medium

E: X-axis scanning end point

L: illumination light

M: pulse motor

Pd: detection position

Pr: position

R1: range

R2: range

R: observation target region

S: scanning start point

W: well

What is claimed is:
 1. A microscope apparatus comprising: an imagingoptical system capable of forming an image of observation target lightindicating an observation target in a container in which the observationtarget is contained, on an imaging element; a drive source that includesa first moving member movable along an optical axis direction of theimaging optical system, and a second moving member movable in theoptical axis direction in a range wider than that of the first movingmember and moves the imaging optical system in the optical axisdirection using the first moving member and the second moving member;and a controller that controls the drive source to cause the imagingelement to form the image of the observation target light in a focusstate when the optical axis reaches a specific region, by moving theimaging optical system in the optical axis direction by using the firstmoving member and the second moving member based on information on adistance in the optical axis direction between the specific region and aregion to be imaged by the imaging element at an imaging position, in acase where it is determined that the specific region exists at aposition of an out-of-focus state in a state where the first movingmember is moved to a limit of a movable range of the first moving memberbefore the optical axis reaches the specific region among respectiveregions, in a state where the imaging element scans the respectiveregions in the container by moving the imaging optical system withrespect to the respective regions in the container by a movement of atleast one of a stage on which the container is installed or the imagingoptical system in a plane intersecting the optical axis direction. 2.The microscope apparatus according to claim 1, wherein an amount ofpower in the optical axis direction distributed by the first movingmember and the second moving member is determined in accordance with arelationship between the information on the distance, information on aposition of the second moving member in the optical axis directionbefore the specific region is scanned, and information on a movingamount of the first moving member.
 3. The microscope apparatus accordingto claim 1, wherein the controller controls the drive source to causethe imaging element to form the image of the observation target light inthe focus state when the optical axis reaches the specific region byapplying the power in the optical axis direction by only the firstmoving member out of the first moving member and the second movingmember to the imaging optical system based on the information on thedistance, in a case where it is determined that the specific regionexists at a position of the focus state in a state where the firstmoving member is moved below the limit of the movable range, before theoptical axis reaches the specific region among the respective regions ina state where the imaging element scans the respective regions in thecontainer.
 4. The microscope apparatus according to claim 3, wherein thepower in the optical axis direction by only the first moving member outof the first moving member and the second moving member is determined inaccordance with a relationship between the information on the distanceand information on a moving amount of the first moving member.
 5. Themicroscope apparatus according to claim 1, wherein the first movingmember is a piezoelectric element that deforms along the optical axisdirection of the imaging optical system.
 6. The microscope apparatusaccording to claim 1, wherein the drive source includes a pulse motor,and the second moving member moves in the optical axis direction byreceiving the power from the pulse motor.
 7. The microscope apparatusaccording to claim 1, further comprising: a detection section that isprovided along the optical axis direction and detects a position of thespecific region in the optical axis direction, wherein the controllercontrols the drive source to cause the imaging element to form the imageof the observation target light in the focus state when the optical axisreaches the specific region by applying the power in the optical axisdirection by the first moving member and the second moving member to theimaging optical system based on the information on the distance, in acase where a position detected by the detection section is the positionof the out-of-focus state in a state where the first moving member ismoved to the limit of the movable range, before the optical axis reachesthe specific region among the respective regions in a state where theimaging element scans the respective regions in the container.
 8. Themicroscope apparatus according to claim 7, wherein the detection sectionincludes a pair of sensors that are provided side by side with theimaging optical system interposed therebetween in a main scanningdirection with respect to the respective regions, and that detect theposition of the specific region in the optical axis directionrespectively, and the controller controls the drive source to cause theimaging element to form the image of the observation target light in thefocus state when the optical axis reaches the specific region byapplying the power in the optical axis direction by the first movingmember and the second moving member to the imaging optical system basedon the information on the distance, in a case where a position detectedby a sensor of the pair of sensors that reaches the specific region inthe main scanning direction earlier is the position of the out-of-focusstate in a state where the first moving member is moved to the limit ofthe movable range, before the optical axis reaches the specific regionamong the respective regions in a state where the imaging element scansthe respective regions in the container.
 9. The microscope apparatusaccording to claim 7, further comprising: a holding member that holdsthe drive source, wherein the detection section is held by the holdingmember.
 10. The microscope apparatus according to claim 1, wherein theimaging optical system has an objective lens movable in the optical axisdirection, and the objective lens is moved in the optical axis directionby the first moving member and the second moving member.
 11. Themicroscope apparatus according to claim 1, wherein the container is awell plate having a plurality of wells.
 12. A non-transitory computerreadable recording medium storing a program causing a computer tofunction as the controller included in the microscope apparatusaccording to claim 1.